Therapeutic proteins are the primary growth driver in the global pharmaceutical market (Kresse, Eur J Pharm Biopharm 72, 479 (2009)). In 2001, biopharmaceuticals accounted for $24.3 billion in sales. By 2007, this number had more than doubled to $54.5 billion. The market is currently estimated to reach $78 billion by 2012 (Pickering, Spectrum Pharmaceutical Industry Dynamics Report, Decision Resources, Inc., 5 (2008)). This includes sales of “blockbuster” drugs such as erythropoietin, tissue plasminogen activator, and interferon, as well as numerous “niche” drugs such as enzyme replacement therapies for lysosomal storage disorders. The unparalleled growth in market size, however, is driven primarily by skyrocketing demand for fully human and humanized monoclonal antibodies (Reichert, Curr Pharm Biotechnol 9, 423 (2008)). Because they have the ability to confer a virtually unlimited spectrum of biological activities, monoclonal antibodies are quickly becoming the most powerful class of therapeutics available to physicians. Not surprisingly, more than 25% of the molecules currently undergoing clinical trials in the United States and Europe are monoclonal antibodies (Reichert, Curr Pharm Biotechnol 9, 423 (2008)).
Unlike more traditional pharmaceuticals, therapeutic proteins are produced in living cells. This greatly complicates the manufacturing process and introduces significant heterogeneity into product formulations (Field, Recombinant Human IgG Production from Myeloma and Chinese Hamster Ovary Cells, in Cell Culture and Upstream Processing, Butler, ed., (Taylor and Francis Group, New York, 2007)). In addition, protein drugs are typically required at unusually high doses, which necessitates highly scalable manufacturing processes and makes manufacturing input costs a major price determinant. For these reasons, treatment with a typical therapeutic antibody (e.g., the anti-HER2-neu monoclonal Herceptin®) costs $60,000-$80,000 for a full course of treatment (Fleck, Hastings Center Report 36, 12 (2006)). Further complicating the economics of biopharmaceutical production is the fact that many of the early blockbuster biopharmaceuticals are off-patent (or will be off-patent soon) and the US and EU governments are expected to greatly streamline the regulatory approval process for “biogeneric” and “biosimilar” therapeutics (Kresse, Eur J Pharm Biopharm 72, 479 (2009)). These factors should lead to a significant increase in competition for sales of many prominent biopharmaceuticals (Pickering, Spectrum Pharmaceutical Industry Dynamics Report, Decision Resources, Inc., 5 (2008)). Therefore, there is enormous interest in technologies which reduce manufacturing costs of protein therapeutics (Seth et al., Curr Opin Biotechnol 18, 557 (2007)).
Many of the protein pharmaceuticals on the market are glycoproteins that cannot readily be produced in easy-to-manipulate biological systems such as bacteria or yeast. For this reason, recombinant therapeutic proteins are produced almost exclusively in mammalian cell lines, primarily Chinese hamster ovary (e.g., CHO-K1), mouse myeloma (e.g., NS0), baby hamster kidney (BHK), murine C127, human embryonic kidney (e.g., HEK-293), or human retina-derived (e.g., PER-C6) cells (Andersen and Krummen, Curr Opin Biotechnol 13, 117 (2002)). Of these, CHO cells are, by far, the most common platform for bioproduction because they offer the best combination of high protein expression levels, short doubling time, tolerance to a wide range of media conditions, established transfection and amplification protocols, an inability to propagate most human pathogens, a paucity of blocking intellectual property, and the longest track record of FDA approval (Field, Recombinant Human IgG Production from Myeloma and Chinese Hamster Ovary Cells, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)).
Large-market biopharmaceuticals are typically produced in enormous stirred-tank bioreactors containing hundreds of liters of CHO cells stably expressing the protein product of interest (Chu and Robinson, Curr Opin Biotechnol 12, 180 (2001), Coco-Martin and Harmsen, Bioprocess International 6, 28 (2008)). Under optimized industrial conditions, such manufacturing processes can yield in excess of 5 g of protein per liter of cells per day (Coco-Martin and Harmsen, Bioprocess International 6, 28 (2008)). Because of the large number of cells involved (˜50 billion cells per liter), the level of protein expression per cell has a very dramatic effect on yield. For this reason, all of the cells involved in the production of a particular biopharmaceutical must be derived from a single “high-producer” clone, the production of which constitutes one of the most time- and resource-intensive steps in the manufacturing process (Clarke and Compton, Bioprocess International 6, 24 (2008)).
The first step in the large-scale manufacture of a biopharmaceutical is the transfection of mammalian cells with plasmid DNA encoding the protein product of interest under the control of a strong constitutive promoter. Stable transfectants are selected by using a selectable marker gene also carried on the plasmid. Most frequently, this marker is a dihydrofolate reductase (DHFR) gene which, when transfected into a DHFR deficient cell line such as DG44, allows for the selection of stable transfectants using media deficient in hypoxanthine. The primary reason for using DHFR as a selectable marker is that it enables a process called “gene amplification”. By growing stable transfectants in gradually increasing concentrations of methotrexate (MTX), a DHFR inhibitor, it is possible to amplify the number of copies of the DHFR gene present in the genome. Because the gene encoding the protein product of interest is physically coupled to the DHFR gene, this results in amplification of both genes with a concomitant increase in the expression level of the therapeutic protein (Butler, Cell Line Development for Culture Strategies: Future Prospects to Improve Yields, in Cell Culture and Upstream Processing, Butler, ed., (Taylor and Francis Group, New York, 2007)). Related systems for the creation of stable bioproduction lines use the glutamine synthetase (GS) or hypoxanthine phosphoribosyltransferase (HPRT) genes as selectable markers and require the use of GS- or HPRT-deficient cell lines as hosts for transfection (Clarke and Compton, Bioprocess International 6, 24 (2008)). In the case of the GS system, gene amplification is accomplished by growing cells in the presence of methionine sulphoximine (MSX) (Clarke and Compton, Bioprocess International 6, 24 (2008)). In the case of the HPRT system, gene amplification is accomplished by growing cells in HAT medium, which contains aminopterin, hypoxanthine, and thymidine (Kellems, ed. Gene amplification in mammalian cells: a comprehensive guide, Marcel Dekker, New York, 1993).
In all of these systems, the initial plasmid DNA comprising a biotherapeutic gene expression cassette and a selectable marker integrates into a random location in the genome, resulting in extreme variability in therapeutic protein expression from one stable transfectant to another (Collingwood and Urnov, Targeted Gene Insertion to Enhance Protein Production from Cell Lines, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). For this reason, it is necessary to screen hundreds to thousands of initial transfectants to identify cells which express acceptably high levels of gene product both before and after gene amplification (Butler, Cell Line Development for Culture Strategies: Future Prospects to Improve Yields, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). A second and more problematic consequence of random gene integration is the phenomenon of transgene silencing, in which recombinant protein expression slows or ceases entirely over time (Collingwood and Urnov, Targeted Gene Insertion to Enhance Protein Production from Cell Lines, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). Because these effects often do not manifest themselves for weeks to months following the initial transfection and screening process, it is generally necessary to carry and expand dozens of independent clonal lines to identify one that expresses the protein of interest consistently over time (Butler, Cell Line Development for Culture Strategies: Future Prospects to Improve Yields, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)).
This large number of screening and expansion steps results in a very lengthy and expensive process to simply generate the cell line that will, ultimately, produce the therapeutic of interest. Indeed, using conventional methods, a minimum of 10 months (with an average of 18 months) and an upfront investment of tens of millions of dollars in labor and material is required to produce an initial pool of protein-expressing cells suitable for industrial manufacturing (Butler, Cell Line Development for Culture Strategies: Future Prospects to Improve Yields, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). If one takes into account lost time on market for a blockbuster protein therapeutic, inefficiencies in cell line production can cost biopharmaceutical manufacturers hundreds of millions of dollars (Seth et al., Curr Opin Biotechnol 18, 557 (2007)).
Much of the time and expense of bioproduction cell line creation can be attributed to random genomic integration of the bioproduct gene resulting in clone-to-clone variability in genotype and, hence, variability in gene expression. One way to overcome this is to target gene integration to a defined location that is known to support a high level of gene expression. To this end, a number of systems have been described which use the Cre, Flp, or ΦC31 recombinases to target the insertion of a bioproduct gene (reviewed in Collingwood and Urnov, Targeted Gene Insertion to Enhance Protein Production from Cell Lines, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). Recent embodiments of these systems, most notably the Flp-In® system marketed by Invitrogen Corp. (Carlsbad, Calif.), couple bioproduct gene integration with the reconstitution of a split selectable marker so that cells with correctly targeted genes can be selected. As expected, these systems result in greatly reduced heterogeneity in gene expression and, in some cases, individual stable transfectants can be pooled, obviating the time and expense associated with expanding a single clone.
The principal drawback to recombinase-based gene targeting systems is that the recombinase recognition sites (IoxP, FRT, or attB/attP sites) do not naturally occur in mammalian genomes. Therefore, cells must be pre-engineered to incorporate a recognition site for the recombinase before that site can be subsequently targeted for gene insertion. Because the recombinase site itself integrates randomly into the genome, it is still necessary to undertake extensive screening and evaluation to identify clones which carry the site at a location that is suitable for high level, long-term gene expression (Collingwood and Urnov, Targeted Gene Insertion to Enhance Protein Production from Cell Lines, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). In addition, the biomanufacturing industry is notoriously hesitant to adopt “new” cell lines, such as those that have been engineered to carry a recombinase site, that do not have a track record of FDA approval. For these reasons, recombinase-based cell engineering systems may not readily be adopted by the industry and an approach that allows biomanufacturers to utilize their existing cell lines is preferable.